1. Field of the Invention
This invention relates to peptide-based antiviral agents and their use. More particularly it concerns peptide-based antiviral agents substantially corresponding in sequence to a region of influenza matrix protein.
2. Description of Prior Work
Influenza viruses, a class of single-stranded RNA virus, continue to cause serious respiratory disease throughout the world. Type A influenza virus causes pneumonia and deaths, especially in the elderly. Type B influenza viruses tend to infect a younger age population than does type A and are associated with Reye's syndrome. With antigenic drift, type B influenza viruses are capable of producing disease in all ages of the population. The economic costs of influenza are considerable: the annual costs of disease due to influenza in the U.S. are estimated to be between 4.6 billion dollars and 10 billion dollars.
Influenza is not a trivial disease. Although a typical influenza case may be limited to fever, sore throat, and several days of malaise with subsequent uneventful recovery, more severe lung disease, primary viral pneumonia, or secondary bacterial pneumonia can occur (Advisory Committee on Immunization Practices, MMWR 35, 317-331 (1986)). The elderly or those who suffer from cardiopulmonary or other chronic lung diseases are at special risk.
The only effective antiviral drugs for influenza are amantadine or its close relative rimantadine. Although these drugs can be quite effective against influenza, they are effective only against disease caused by type A influenza virus and are not well tolerated in the group of individuals at highest risk of morbidity and mortality--the elderly. In individuals with poor renal clearance, the drugs may accumulate, producing convulsion; other CNS effects are light-headedness, dizziness, and problems in concentrating (id.; and Dolin and Bentley in "Options for the Control of Influenza," Kenda and Patriarca (eds.), Alan R. Liss, Inc., New York 1986, pp. 317-326). In addition, the drug works most effectively as a prophylactic agent; therefore, one risks side effects in the absence of actual infection with influenza. Considerable morbidity and mortality also occur with type B influenza, which was responsible for the major epidemic illness in four out of the last ten influenza epidemics in the United States (MMWR 35, 470-479 (1986)).
The present invention provides an antiviral agent that functions by targeting the viral transcription. These agents are not only specific for influenza virus, but are also free of the antigenic shift and drift associated with the surface antigens of influenza virus, i.e., hemagglutinin and neuraminidase. Thus these antiviral agents have a broad spectrum, inhibiting the transcription of type A as well as type B influenza viruses, with possible extension to the RNA polymerases of other negative-strand viruses, including the paramyxovirus and rhabdovirus groups responsible for human and veterinary disease.
M.sub.1 ("matrix protein") is a major structural component of the influenza virion, constituting approximately 30% of the total viral protein and occupying the key location between the surface glycoprotein of the envelope and the ribonucleoprotein complex (Virology 42, 890-904 (1979)). M.sub.1 incorporates into lipid bilayers either as liposomes or planar bilayer lipid membranes (Bucher, D. J. et al., J. Virol. 36, 586-590 (1980); Bucher, D. J. et al., Intervirology 14, 69-77 (1988); and Kahn, M. W. et al., J. Clin. Microbiol. 16, 115-122 (1982)).
M.sub.1 has been shown to inhibit influenza virus transcription, and this activity has been shown to be localized in the 15 kd amino terminal fragment (Zavonarjev and Ghendon, J. Virol. 33, 583-586 (1980); and Ye, Z. et al., J. Virol. 61, 239-246 (1987)). This effect can be reversed by monoclonal antibodies (Hankins, R. W. et al., Virus Genes 3, 111-126 (1989)). Ye, Z. et al., J. Virol. 63, 3586-3594 (1989), studied the transcription inhibition and determined the RNA binding domains using anti-idiotypic antibodies and synthetic peptides. As background, we performed immunofluorescence analysis of M.sub.1 with monoclonal antibodies (MAbs) and observed the migration of M.sub.1 to the nucleus during the replicative cycle and the association of M.sub.1 with actin filaments in the cytoplasm (Bucher, D. et al., J. Virol. 63, 3622-3633 (1989)). M.sub.1 is highly conserved, unlike the highly mutable surface antigens hemagglutinin and neuraminidase. Comparison of the amino acid sequence of M.sub.1 from influenza strain A/PR/8/34 and strain A/Udorn/72 shows only seven amino acids changed over a period of 38 years (Winter and Fields, Nucleic Acids Res. 8, 1965-1974 (1980; and Lamb and Lai, Virology 112, 746-751 (1981)). Furthermore, these changes are conservative, including such alterations as Ile.fwdarw.Ala, and Arg.fwdarw.Lys. Antigenic drift in hemagglutinin occurs at a rate of 0.9-1% of the amino acids/year within a subtype, as seen for A/NT/60/68 versus A/Bangkok/79 strains (Huddleston and Brownlee, Nucleic Acids Res. 10, 1029-1038 (1982)). Overall sequence homology between M.sub.1 of type A and type B is found to be 54%; however, in certain regions there is more than 70% homology. Thus, it is likely that a peptide antiviral with a broad spectrum of activity (both A and B types) will result if it incorporates conserved sequences present in M.sub.1 with virus transcription inhibitory activity.
Other related work with M.sub.1 protein has demonstrated that M.sub.1 will incorporate into lipid bilayers liposomes or planar membranes (Bucher 1980, supra). Antibody response to the M.sub.1 component in a clinical population that was immunized with influenza vaccine or infected with wild-type circulating virus has been studied (Khan, M. W. et al., J. Clin. Microbiol. 16, 813 (1982)). It has been demonstrated that M.sub.1 can be an effective target for universal detection of type A influenza viruses in clinical specimens (Bucher, D. J. et al., J. Immunol. Methods 96, 77-85 (1987)). A panel of monoclonal antibodies to several antigenic sites of M.sub.1 has been developed and used in virus detection (id.; and Bucher, D. et al., VIIth International Congress of Virology, Edmonton, Canada, Abstract, R2328). We have localized three immunoreactive segments of M.sub.1 using synthetic peptides (Bucher 1989, supra; and U.S. Pat. No. 4,981,782).
While these earlier studies have provided valuable insight into the mechanism and potential prevention of influenza infection through immunization, an important need remains for agents which will intervene in the disease by direct antiviral action. It is this need that the present invention addresses.
References
The following references were collected by the inventors and relate to the general subject of influenza, influenza virus, antiviral activity of peptides and the like. Many of the references are cited throughout this document.
1. National Academy of Sciences. New vaccine development. Establishing priorities. Vol. I. Diseases of Importance in the United States, Washington, D.C., National Academy Press, Appendix K, Prospects for immunizing against influenza viruses A and B (1985). PA1 2. Advisory Committee on Immunization Practices (ACIP). Prevention and control of influenza. MMWR 35, 317-331 (1986). PA1 3. R. Dolin and D. W. Bentley. Amantadine and rimantadine: prophylaxis and therapy of influenza A in adults. In "Options for the Control of Influenza. A. P. Kenda and P. A. Patriarca (eds.). Alan R. Liss, Inc., New York 1986, pp. 317-326. PA1 4. Centers for Disease Control (CDC). Influenza-United States, 1985-1986 season. MMWR 35, 470-479 (1986). PA1 5. I. T. Schulze. The structure of influenza virus. I. The polypeptide of the virion. Virology 42, 890-904 (1979). PA1 6. D. J. Bucher, I. G. Kharitonenkov, J. A. Zakomirdin, V. B. Gregoriev, S. M. Klimenko, and J. F. Davis. Incorporation of influenza virus M-protein into liposomes. J. Virol. 36, 586-590 (1980). PA1 7. D. J. Bucher, I. G. Kharitonenkov, D. K. Lvov, T. V. Pysine, and H. M. Lee. Comparative study of influenza virus H.sub.2 (Asian) heamgglutinins isolated from human and avian sources. Intervirology 14 69-77 (1988). PA1 8. M. W. Khan, M. Gallagher, D. Bucher, C. P. Cerini, and E. D. Kilbourne. Detection of influenza virus neuraminidase-specific antibodies by an enzyme-linked immunosorbent assay. J. Clin. Microbiol. 16, 115-122 (1982). PA1 9. A. Y. Zavonarjev and Y. Z. Ghendon. Influence of membrane (M) protein on influenza A virus virion transcript activity in vitro and its susceptibility to rimantadine. J. Virol. 33, 583-586 (1980). PA1 10. Z. Ye, R. Pal, J. W. Fox, and R. R. Wagner. Functional and antigenic domains of the matrix (M) protein of influenza A virus. J. Virol. 61, 239-246 (1987). PA1 11. R. W. Hankins, K. Nagata, D. J. Bucher, S. Popple, and A. Ishihama. Monoclonal antibody analysis of influenza virus matrix protein epitopes involved in transcription inhibition. Virus Genes 3, 111-126 (1989). PA1 12. Z. Ye, N. W. Baylor, and R. R. Wagner. Transcription-inhibition and RNA-binding domains of influenza A virus matrix protein mapped with anti-idiotypic antibodies and synthetic peptides. J. Virol. 63, 3586-3594 (1989). PA1 13. D. Bucher, S. Popple, M. Baer, A. Mikhail, Y. -F. Gong, C. Whiteker, E. Paoletti, and A. Judd. M protein (M1) of influenza virus: Antigenic analysis and intracellular localization with monoclonal antibodies. J. Virol. 63, 3622-3633 (1989). PA1 14. G. Winter and S. Fields. Cloning of influenza DNA into M1. The sequence of the RNA segment encoding the A/PR/8/34 matrix protein. Nucleic Acids Res. 8, 1965-1974 (1980). PA1 15. R. A. Lamb and C. J. Lai. Conservation of the influenza virus membrane protein (M1) amino acid sequence and an open reading frame of RNA segment 7 encoding a second protein (M2) in H1N1 and H3N3 strains. Virology 112,746-751 (1981). PA1 16. J. A. Huddleston and G. G. Brownlee. The sequence of the nucleoprotein gene of human influenza A virus strain A/NT/60/68. Nucleic Acids Res. 10, 1029-1038 (1982). PA1 17. D. J. Bucher. Chromatographic isolation of the major polypeptides of influenza virus. In The Negative Strand Viruses. B. W. J. Mahy and R. D. Barry (eds.), Vol. I, Academic Press, 1975, pp. 133-143. PA1 18. D. J. Bucher, S. S. -L. Li, J. M. Kehoe, and E. D. Kilbourne. Chromatographic isolation of the hemagglutinin polypeptides from influenza virus vaccine and determination of their amino terminal sequences. Proc. Natl. Acad. Sci. USA 73, 238-242 (1976). PA1 19. D. J. Bucher. Purification of neuraminidase from influenza viruses by affinity chromatography. Biochim. Biophys. Acta 483, 393-399 (1977). PA1 20. M. Gallagher, D. J. Bucher, R. Dourmashkin, J. F. Davis, G. Rosenn, and E. D. Kilbourne. Isolation of immunogenic neuraminidases of human influenza viruses by a combination of genetic and biochemical procedures. J. Clin. Microbiol. 20, 89-93 (1984). PA1 21. M. W. Khan, D. J. Bucher, A. K. Koul, G. Kalish, and E. D. Kilbourne. Detection of antibodies to influenza virus M protein by an enzyme-linked immunosorbent assay. J. Clin. Microbiol. 16, 813 (1982). PA1 22. D. J. Bucher, I. G. Kharitonenkov, M. W. Khan, A. Palo, D. Holloway, and A. Mikhail. Detection of influenza viruses through selective adsorption and detection of the M-protein antigen. J. Immunol. Methods 96, 77-85 (1987). PA1 23. D. Bucher, A. Mikhail. S. Popple, M. Baer, and C. Whitaker. Rapid detection of influenza viruses with monoclonal antibodies to M-protein. VIIth International Congress of Virology, Edmonton, Canada, Abstract, R2328. PA1 24. A. K. Judd, D. J. Bucher, and S. W. Popple. Synthetic peptides for diagnosis and prevention of influenza virus infection and their use. U.S. Pat. No. 4,981,782, Jan. 1, 1991. PA1 25. B. W. Erickson and R. B. Merrifield. Solid phase peptide synthesis. In The Proteins, vol. II. H. Neurath (ed.), Academic Press, Inc., New York, pp. 255-527 (1976). PA1 26. D. J. Bucher, I. G. Kharitonenkov, J. A. Zakomirdin, V. B. Gregoriev, S. M. Klimenko, and J. F. Davis. Incorporation of influenza virus M-protein into liposomes. J. Virol. 36, 586-590 (1980). PA1 27. O. M. Rochovansky. RNA synthesis by ribonucleoprotein-polymerase complexes isolated from influenza virus. Virology 73, 327-338 (1976). PA1 28. J. J. Plotch, M. Bouloy, I. Ulmann, and R. M. Krug. A unique cap (M.sup.7 GippXm) dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral transcription. Cell 23, 847-858 (1981). PA1 29. A. Kato, K. Mizumoto, and A. Ishihama. Purification and enzymatic properties of an RNA polymerase -RNA complex from influenza virus. Virus Res. 3, 115-127 (1985). PA1 30. R. G. Almquist, W. -R. Chao, A. K. Judd, C. Mitoma, D. J. Rossi, R. E. Panasevich, and R. J. Matthews. Synthesis and biological activity of ketomethylene-containing monopeptide analogs of snake venom angiotensin converting enzyme inhibitors. J. Med. Chem. 31, 561 (1988). PA1 31. J. M. Stewart and J. D. Young. In Solid Phase Peptide Synthesis. J. M. Stewart and J. D. Young (eds.). Pierce Chemical Co., 1984, p. 83. PA1 32. S. L. Harbeson and D. H. Rich. Inhibition of aminopeptidases by peptides containing ketomethylene and hydroxyethylene amide bond replacements. J. Med. Chem. 32, 1378-1392 (1989). PA1 33. M. T. Garcia-Lopez, R. Gonzalez-Muniz, and J. R. Harto. Synthesis of ketomethylene dipeptides containing basic amino acid analogs of C-terminus. Tetrahedron 44, 5131-5138 (1988). PA1 34. M. T. Garcia-Lopez, R. Gonzalez-Muniz, and J. R. Harto. A simple and versatile route to ketomethylene dipeptide analogs. Tetrahedron Lett. 29, 1577-1580 (1988). PA1 35. R. L. Johnson and R. B. Miller. Use of triphenylmethyl (trityl) amino protecting group in the synthesis of ketomethylene analogs of peptides. Int. J. Peptide Protein Res. 23, 581-590 (1984). PA1 36. J. V. N. V. Prasad and D. H. Rich. Addition of allylic metals for .alpha.-amino aldehydes. Application to the synthesis of statin, ketomethylene and hydroethylene dipeptide diesters. Tetrahedron Lett. 31, 1803-1806 (1990). PA1 37. B. E. Evans, K. E. Rittle, C. F. Homnick, J. P. Springer, J. Hirshfield, and D. F. Veber. A stereocontrolled synthesis of hydroxyethylene dipeptide isosteres using novel, chiral aminoalkyl epoxides and .gamma.-(aminoalkyl) .gamma.-lactones. J. Org. Chem. 50, 4615-4625 (1985). PA1 38. A. H. Fray, R. L. Kaye, and E. F. Kleinman. A short stereoselective synthesis of the lactone precursor to 2R,4S,5S hydroxyethylene dipeptide isosteres. J. Org. Chem. 51, 4828-4833 (1986). PA1 39. K. Deres, H. Schild, K. -H., Wiesmuller, G. Jung, and H. -G. Rammensee. In vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine. Nature 342, 561-564 (1989). PA1 40. I. Toth, R. A. Hughes, M. R. Munday, P. Mascagni, and W. A. Gibbons. A novel oligopeptide delivery system for poorly adsorbed peptides and drugs. In Proceedings of the Eleventh American Peptide Symposium. J. E. Rivier and G. R. Marshall (eds.), ESCOM, Leiden, pp. 1078-1079 (1990). PA1 41. Y. Sanchez, I. Ionescu-Matiu, G. R. Dreesman, W. Kramp, H. R. Six, F. B. Hollinger, and J. L. Melnick. Humoral and cellular immunity to hepatitis B virus-derived antigens: comparative activity of Freund's complete adjuvant, alum, and liposomes. Infect. Immun. 30, 728-733 (1980). PA1 42. G. W. Both and G. M. Air. Nucleotide sequence coding for the N-terminal region of the matrix protein of influenza virus. Eur. J. Biochem. 96, 363-372 (1970). PA1 43. B. E. Johansson, D. J. Bucher, and E. D. Kilbourne. Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection. J. Virol. 63, 1239-1246 (1989). PA1 44. A. Gregorriades. Influenza virus-induced proteins in nuclei and cytoplasm of infected cells. Virology 79, 449-454 (1977).